Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrow–derived mesenchymal stromal cells

Background aimsA medium supplemented with fetal bovine serum (FBS) is of common use for the expansion of human mesenchymal stromal cells (MSCs). However, its use is discouraged by regulatory authorities because of the risk of zoonoses and immune reactions. Human platelet lysate (PL) obtained by freezing/thawing disruption of platelets has been proposed as a possible substitute of FBS. The process is time-consuming and not well standardized. A new method for obtaining PL that is based on the use of ultrasound is proposed.MethodsPlatelet sonication was performed by submerging platelet-containing plastic bags in an ultrasonic bath. To evaluate platelet lysis we measured platelet-derived growth factor-AB release. PL efficiency was tested by expanding bone marrow (BM)-MSCs, measuring population doubling time, differentiation capacity and immunogenic properties. Safety was evaluated by karyotyping expanded cells.ResultsAfter 30 minutes of sonication, 74% of platelet derived growth factor-AB was released. PL enhanced BM-MSC proliferation rate compared with FBS. The mean cumulative population doubling (cPD) of cells growth in PL at 10%, 7.5% and 5% was better compared with cPD obtained with 10% FBS. PD time (hours) of MSCs with PL obtained by sonication was shorter than for cPD with PL obtained by freezing/thawing (18.9 versus 17.4, P < 0.01). BM mononucleated cells expressed MSC markers and were able to differentiate into adipogenic, osteogenic and chondrogenic lineages. When BM-MSCs and T cells were co-cultured in close contact, immunosuppressive activity of BM-MSCs was maintained. Cell karyotype showed no genetic alterations.ConclusionsThe proposed method for the production of PL by sonication could be a safe, efficient and fast substitute of FBS, without the potential risks of FBS.     

Chloroplast biogenesis of photosystem II cores involves a series of assembly-controlled steps that regulate translation

The biogenesis of photosystem II, one of the major photosynthetic protein complexes, involves a cascade of assembly-governed regulation of translation of its major chloroplast-encoded subunits. In Chlamydomonas reinhardtii, the presence of the reaction center subunit D2 is required for the expression of the other reaction center subunit D1, while the presence of D1 is required for the expression of the core antenna subunit apoCP47. Using chimeric genes expressed in the chloroplast, we demonstrate that the decreased synthesis of D1 or apoCP47 in the absence of protein assembly is due to a genuine downregulation of translation. This regulation is mediated by the 5' untranslated region of the corresponding mRNA and originates from negative feedback exerted by the unassembled D1 or apoCP47 polypeptide. However, autoregulation of translation of subunit D1 is not implicated in the recovery from photoinhibition, which involves an increased translation of psbA mRNA in response to the degradation of photodamaged D1. De novo synthesis and repair of photosystem II complexes are independently controlled.     

5′‐ectonucleotidase mediates multiple‐drug resistance in glioblastoma multiforme cells

Glioblastoma multiforme (GBM) cells are characterised by their extreme chemoresistance. The activity of multiple‐drug resistance (MDR) transporters that extrude antitumor drugs from cells plays the most important role in this phenomenon. To date, the mechanism controlling the expression and activity of MDR transporters is poorly understood. Activity of the enzyme ecto‐5′‐nucleotidase (CD73) in tumor cells, which hydrolyses AMP to adenosine, has been linked to immunosuppression and prometastatic effects in breast cancer and to the proliferation of glioma cells. In this study, we identify a high expression of CD73 in surgically resected samples of human GBM. In primary cultures of GBM, inhibition of CD73 activity or knocking down its expression by siRNA reversed the MDR phenotype and cell viability was decreased up to 60% on exposure to the antitumoral drug vincristine. This GBM chemosensitization was caused by a decrease in the expression and activity of the multiple drug associated protein 1 (Mrp1), the most important transporter conferring multiple drug resistance in these cells. Using pharmacological modulators, we have recognized the adenosine A₃ receptor subtype in mediation of the chemoresistant phenotype in these cells. In conclusion, we have determined that the activity of CD73 to trigger adenosine signaling sustains chemoresistant phenotype in GBM cells. J. Cell. Physiol. 228: 602–608, 2013. © 2012 Wiley Periodicals, Inc.     

Full-thickeness anterior blepharotomy and transpalpebral fat decompression in Graves' orbitopathy

A chief morbidity of Graves eye disease is eyelid retraction and exophthalmus. Transpalpebral orbital fat removal accomplished with full thickness anterior blepharotomy was performed in 4 patients (5 orbits). Preoperative and postoperative ocular exposure symptoms, visual acuity, upper eyelid retraction and proptosis were evaluated. In all 5 operated orbits preoperative symptoms resolved; good results were achieved from the functional and cosmetic point of view. Full-thickness anterior blepharotomy combined with fat decompression is a safe and effective surgery for patients with upper eyelid retraction and exophthalmus due to enlarged orbital fat compartment.     

Evidence of involvement of leptin and IL-6 peptides in the action of interferon-beta in secondary progressive multiple sclerosis

Leptin is a peptide hormone which acts on cells of immune system by influencing the production of cytokines. Serum leptin levels and cytokine production by peripheral blood mononuclear cells (PBMC) were measured in 18 secondary progressive multiple sclerosis (SPMS) patients under IFN-beta-1b treatment. There were no overall effects on leptin, interleukin-6 (IL-6), IL-10 and IL-12 p40 after 2, 6 and 12 months of treatment. However, leptin and IL-6 decreased after 6 and 12 months of treatment in 12 patients who did not show progression of disability. Thus, our pilot data show that the beneficial effect of IFN-beta on some SPMS patients might be associated with the reduced levels of leptin and reduced IL-6 production by PBMC.     

Multiscale modeling of the cardiovascular system: application to the study of pulmonary and coronary perfusions in the univentricular circulation

The objective of this study is to compare the coronary and pulmonary blood flow dynamics resulting from two configurations of systemic-to-pulmonary artery shunts currently utilized during the Norwood procedure: the central (CS) and modified Blalock Taussig (MBTS) shunts. A lumped parameter model of the neonatal cardiovascular circulation and detailed 3-D models of the shunt based on the finite volume method were constructed. Shunt sizes of 3, 3.5 and 4 mm were considered. A multiscale approach was adopted to prescribe appropriate and realistic boundary conditions for the 3-D models of the Norwood circulation. Results showed that the average shunt flow rate is higher for the CS option than for the MBTS and that pulmonary flow increases with shunt size for both options. Cardiac output is higher for the CS option for all shunt sizes. Flow distribution between the left and the right pulmonary arteries is not completely balanced, although for the CS option the discrepancy is low (50-51% of the pulmonary flow to the right lung) while for the MBTS it is more pronounced with larger shunt sizes (51-54% to the left lung). The CS option favors perfusion to the right lung while the MBTS favors the left. In the CS option, a smaller percentage of aortic flow is distributed to the coronary circulation, while that percentage rises for the MBTS. These findings may have important implications for coronary blood flow and ventricular function.     

Evidence for a constitutive cytoplasmic cutinase in ungerminated conidia of Botrytis cinerea Pers.: Fr.

Cytoplasmic soluble proteins from ungerminated conidia of Botrytis cinerea exhibited cutinase activity, while cell wall binding proteins lacked this activity. Cutinase activity in proteins extracted from cell walls and cytoplasm of ungerminated conidia of Botrytis cinerea was determined using p-nitrophenyl butyrate (PNB) and TLC analysis of products derived from hydrolysis of [³H]cutin. Treatment of conidia with indoxyl acetate, a substrate indicative of non-specific esterase and cutinase activity, also gave a positive reaction in the cytoplasm of ungerminated conidia. The possible role of a putative constitutive cutinase in the cytoplasm of conidia in the early stages of infection of plants by B. cinerea is discussed.     

A poised chromatin platform for TGF-β access to master regulators

Specific chromatin marks keep master regulators of differentiation silent yet poised for activation by extracellular signals. We report that nodal TGF-β signals use the poised histone mark H3K9me3 to trigger differentiation of mammalian embryonic stem cells. Nodal receptors induce the formation of companion Smad4-Smad2/3 and TRIM33-Smad2/3 complexes. The PHD-Bromo cassette of TRIM33 facilitates binding of TRIM33-Smad2/3 to H3K9me3 and H3K18ac on the promoters of mesendoderm regulators Gsc and Mixl1. The crystal structure of this cassette, bound to histone H3 peptides, illustrates that PHD recognizes K9me3, and Bromo binds an adjacent K18ac. The interaction between TRIM33-Smad2/3 and H3K9me3 displaces the chromatin-compacting factor HP1γ, making nodal response elements accessible to Smad4-Smad2/3 for Pol II recruitment. In turn, Smad4 increases K18 acetylation to augment TRIM33-Smad2/3 binding. Thus, nodal effectors use the H3K9me3 mark as a platform to switch master regulators of stem cell differentiation from the poised to the active state.     

The targeting of plasmalemmal ceramide to mitochondria during apoptosis

Ceramide is a key lipid mediator of cellular processes such as differentiation, proliferation, growth arrest and apoptosis. During apoptosis, ceramide is produced within the plasma membrane. Although recent data suggest that the generation of intracellular ceramide increases mitochondrial permeability, the source of mitochondrial ceramide remains unknown. Here, we determine whether a stress-mediated plasmalemmal pool of ceramide might become available to the mitochondria of apoptotic cells. We have previously established annexin A1--a member of a family of Ca(2+) and membrane-binding proteins--to be a marker of ceramide platforms. Using fluorescently tagged annexin A1, we show that, upon its generation within the plasma membrane, ceramide self-associates into platforms that subsequently invaginate and fuse with mitochondria. An accumulation of ceramide within the mitochondria of apoptotic cells was also confirmed using a ceramide-specific antibody. Electron microscopic tomography confirmed that upon the formation of ceramide platforms, the invaginated regions of the plasma membrane extend deep into the cytoplasm forming direct physical contacts with mitochondrial outer membranes. Ceramide might thus be directly transferred from the plasma membrane to the mitochondrial outer membrane. It is conceivable that this "kiss-of-death" increases the permeability of the mitochondrial outer membrane thereby triggering apoptosis.     

Electroencephalographic Patterns in Chronic Pain: A Systematic Review of the Literature

The main objective of this study is to review and summarize recent findings on electroencephalographic patterns in individuals with chronic pain. We also discuss recent advances in the use of quantitative Electroencephalography (qEEG) for the assessment of pathophysiology and biopsychosocial factors involved in its maintenance over time. Data collection took place from February 2014 to July 2015 in PubMed, SciELO and PEDro databases. Data from cross-sectional studies and longitudinal studies, as well as clinical trials involving chronic pain participants were incorporated into the final analysis. Our primary findings related to chronic pain were an increase of theta and alpha EEG power at rest, and a decrease in the amplitude of evoked potentials after sensory stimulation and cognitive tasks. This review suggests that qEEG could be considered as a simple and objective tool for the study of brain mechanisms involved in chronic pain, as well as for identifying the specific characteristics of chronic pain condition. In addition, results show that qEEG probably is a relevant outcome measure for assessing changes in therapeutic studies.